Development of a refined tenocyte differentiation culture technique for tendon tissue engineering.

We have established that human tenocytes can differentiate in the absence of exogenous fetal bovine serum (FBS) but in the presence of insulin-like growth factor-1 (IGF-1) and transforming growth factor-β3 (TGF-β3). The extent of tenocyte differentiation was assessed by examining cell survival, collagen synthesis, cell morphology and expression of tenocyte differentiation markers such as scleraxis (Scx), tenomodulin (Tnmd), collagen type I (Col-I) and decorin (Dcn). Our results indicate that 50 ng/ml IGF-1 and 10 ng/ml TGF-β3 (in the absence of FBS) were capable of maintaining in vitro human tenocyte survival in 14-day cultures. The extent of collagen synthesis and messenger ribonucleic acid expression of Scx, Tnmd, Col-I and Dcn were significantly upregulated in response to IGF-1 and TGF-β3. These findings have shown for the first time that human tenocytes can be maintained in long-term culture, in serum-free conditions, making this approach a suitable one for the purpose of tendon tissue engineering.